The invention pertains to the preparation and use of thin, photobleachable luminescent layers for calibration and standardization of optical imaging devices, such as optical or Raman microscopy. For the quantitative application of optical and Raman microscopy, it is essential that the intensities in the images acquired with these microscopic techniques are determined only by the spatial distribution of the concentration, absorption, and emission characteristics of the luminophores in the specimen under investigation. If this is not possible, the image intensities should at least be proportional to these parameters. Generally, however, image intensity variations are not only determined by the specimen, but also by spatial non-uniformities of the optical system of the microscope across the field of view, so that only qualitative investigations can be performed. In order to realize the images required for quantitative microscopy, the microscope must be calibrated and standardized. The thus obtained images allow the comparison of different samples obtained on the same microscope, also at a different point in time, or the comparison of images obtained on different microscopes, provided that the different microscopes have been calibrated in the same way.
In earlier work, calibration and standardization of an optical microscope was attempted by an approach using images of a uniform luminescent layer (K. R. Castleman, Digital Image Processing. Prentice-Hall, Englewood Cliffs, N.J., 1979, and Z. Jericevic, B. Wiese, J. Brayan & L. C. Smith, "Validation of an Image System," in Luminescence Microscopy of Living Cells in Culture, Part B, Quantitative Luminescence Microscopy-Imaging and Spectroscopy, edited by D. Lansing Taylor and Y. Wang, Academic Press, San Diego, Calif., 1989). Such an approach has three disadvantages. Firstly, in the case of an image of a luminescent layer, the product of the illumination and detection efficiency distributions is measured, and no information on the separate distributions is available. Secondly, completely uniform luminescent layers are difficult to obtain. Thirdly, the results of calibration and standardization based on this approach are affected by luminescence photobleaching of the layer. For general calibration and standardization in optical microscopy, it would be preferable to have an approach which does not suffer from these disadvantages. Jericevic et al. (Z. Jericevic, B. Wiese, J. Brayan & L. C. Smith, "Validation of an Image System," in Fluorescence Microscopy of Living Cells in Culture, Part B, Quantitative Fluorescence Microscopy-Imaging and Spectroscopy, edited by D. Lansing Taylor and Y. Wang, Academic Press, San Diego, Calif., 1989) attempted to do away with the first disadvantage by using luminescence photobleaching techniques for the determination of only the illumination distribution. In his method, at least 20 images of a uniform, photobleaching luminescent layer were required. They showed that by numerically fitting the luminescence intensity decrease in each pixel of the first image with an exponential function, it was possible to determine only the excitation intensity distribution in the field of view of the used microscope (Z. Jericevic, D. M. Benson, J. Bryan, & L. C. Smith, "Rigorous Convergence Algorithm for Fitting a Monoexponential Function with a Background Term Using the Least-Squares Method," Anal. Chem., 59 (1987), 658-662). There are several drawbacks to this method and experimental approach. Firstly, a luminescent layer has to be prepared by spreading an FITC-IgG mixture on a microscope slide. With such a procedure, it is very difficult to obtain a uniform luminescent layer. Secondly, the method provides only the illumination distribution; no information about the detection distribution is obtained. Thirdly, the determination of the illumination distribution is based on numerically fitting routines, which renders the method relatively slow.